Joseph A. Babitch

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.

Synaptic Plasma Membrane Tubulin May Be an Integral Constituent

Abstract: Mild detergent extraction of chick brain synaptic plasma membranes followed by gel electrophoresis suggests that synaptic plasma membrane tubulin is an integral component. Although some of the synaptic plasma membrane tubulin might be aggregates, that possibility is not supported by the observation that tubulin aggregates that are added to synaptosomes before synaptic subfractionation do not partition with synaptic plasma membranes during membrane isolation.

Identification of myosin in isolated synaptic junctions

A monospecific antibody prepared against chicken gizzard myosin reacted with only one peptide corresponding to myosin heavy chain (Mr = 200,000) in gels of synaptic plasma membranes (SPM) and synaptic junctions (SJ) prepared from several species. Preadsorption of antisera with purified brain myosin eliminated antibody reactivity to SPMs and SJs. SJs were found to contain approximately 3 times the concentration of myosin found in SPMs when assayed by an indirect immunoradiometric assay. Postsynaptic density and myelin fractions contained no myosin detectable by immunoradiometric assay, antibody binding to gels, or Coomassie blue staining. The band identified as myosin in SJ fraction yielded peptide fingerprints indistinguishable from fingerprints of purified brain myosin but distinct from fingerprints of purified smooth and skeletal muscle myosins. The distribution of exogenous [125I]myosin during subcellular fractionation indicated that myosin in isolated synaptic junction could not have resulted from artifactual re-distribution of soluble myosin. Together these results show that a non-muscle myosin is an endogenous component of CNS asymmetric synapses.

Composition of an isoelectric focusing gel yielding a broad pH gradient

Abstract An isoelectric focusing gel system is described which produces a pH gradient spanning the range 4–9. When chick brain mitochondrial polypeptides were focused on such a gel, extra polypeptide spots were observed in the basic region which were not seen in a gel prepared by conventional methods.

Identification and comparison of protein I in chick and rat forebrain

Abstract: Protein I has been identified and compared in membranes prepared from chick and rat forebrain. Based upon five criteria known to characterize protein I, namely, (1) its ability to serve as a substrate for both the cyclic AMP‐dependent protein kinase and (2) the Ca2+‐dependent, calmodulin‐requiring protein kinase, (3) its ability to be extracted from membranes at low pH, (4) its characteristic pattern of digestion by collagenase, and (5) its existence as a basic protein, we have determined that although protein I of rat brain consists of the usual doublet polypeptides la and Ib, only a single chick forebrain polypeptide is detectable which possesses protein Mike properties.

Application of bradford's protein assay to chick brain subcellular fractions

Protein content of different subcellular fractions from chick brain is compared by using Lowry, TCA-Lowry and Bradford assay methods. Caution is urged in application of Bradfords method to general assay for protein concentration in subcellular fractions.

Topography of glycoproteins in the chick synaptosomal plasma membrane

Chick brain synaptosomes or synaptic subfractions were treated with neuraminidase (EC 3.2.1.18) and/or galactose oxidase (EC 1.1.3.9) preparations in which proteolytic activity was inhibited with phenylmethanesulfonyl fluoride followed, after washing, by reductive incorporation of sodium boro[3H]hydride to identify galactose residues exposed on the synaptosomal external surface. Control experiments to demonstrate restriction of labeling to the external surface involved comparing the radioactivity in synaptoplasmic, soluble polypeptides isolated after labeled, isolated synaptoplasm and examining incorporation into fractions incubated without enzymes. Intactness of the synaptic plasma membrane after labeling was shown by trypsin digestion studies. Polypeptides were separated on sodium dodecyl sulfate polyacrylamide gels and were detected by a liquid scintillation counting procedure. Eleven major radioactive peaks were found after galactose oxidase treatment and reduction of isolated synaptic membranes. When intact synaptosomes were labeled, the same components were detected. When isolated synaptic membranes or intact synaptosomes were treated with neuraminidase before galactose oxidase treatment, three additional components were labeled. These results suggest that (a) chick synaptic membranes have a complex mixture of glycoproteins, (b) all major chick synaptic membrane glycoproteins labeled by galactose oxidase have most or all carbohydrate groups exposed at the exterior surface of the synaptosome, (c) all major, externally-disposed polypeptides of these synaptic membranes are glycoproteins.

Calcium binding to tubulin

Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites (1.56 +/- 0.38 per tubulin dimer) with a dissociation constant of (4.86 +/- 0.12).10(-6) M and low-affinity binding sites (5.82 +/- 0.50 per tubulin dimer) with a dissociation constant of (6.4 +/- 0.4).10(-5) M. In the presence of 6.10(-5) M MgSO4, we found 0.64 +/- 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of (4.7 +/- 0.5).10(-6) M and 1.2 +/- 0.2 sites per dimer with a dissociation constant of (3.5 +/- 0.4).10(-5) M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium-binding sites.

Protein-load effects on the pH gradient of isoelectric focusing in polyacrylamide gels

Abstract The pH of the most basic section of an isoelectric-focusing gradient prepared in a polyacrylamide gel decreases in direct proportion to the amount of chick or rat liver or brain protein focused in the gel. At modest protein loads (0.3 mg) the decrease in pH is relatively small (0.28 pH unit) but at high protein loads the decrease in pH becomes appreciable (∼ 1.00 pH unit). This phenomenon is independent of the source of the ampholytes. To observe basic proteins in two-dimensional, equilibrium polyacrylamide gels low protein loads combined with silver staining is recommended.

Calcium-binding proteins in whole brain and synaptic subfractions

At least 19 calcium-binding proteins were detected in avian brain subfraction using 45Ca2+ binding to proteins immobilized in polyacrylamide gels. Half of the 45Ca2+ binding proteins were observed in presynaptic cytoplasm. Two-dimensional gel electrophoresis of this material revealed at least 14 45Ca2+ binding polypeptides besides calmodulin. These proteins may be important in brain and nerve terminal function.